Wednesday, 25 July, 2018

It’s getting hot in here, why don’t you change your twist

Biophysics

Single-molecule magnetic tweezers set-up. Picture: F. Kriegel

Increasing temperature changes the twist of the DNA helix. To quantify this process, an international team of researchers led by the biophysicist Prof Jan Lipfert applied single-molecule magnetic tweezers measurements and extensive computer simulations to DNA molecules.

DNA is the carrier of all cellular genetic information and increasingly used as construction material in nanotechnology. Applying new methods, DNA structures can be precisely build up. Crucial basis for prediction of performance and stability of such designed structures is the knowledge about the conformations and (mechanical) properties of DNA. A defining feature is its helicity – but one has to be aware of the natural unwinding of the DNA helix with increasing temperatures (much lower than its melting temperature).This property was not well characterized so far, hence, Professor Jan Lipfert and collaborators quantitatively characterized the temperature dependency of DNA twist and the structural changes after unwinding the DNA to by some degrees upon using single-molecule magnetic tweezers measurements, and atomistic molecular dynamics and coarse-grained simulations. The results are presented in Nucleic Acids Research.

The DNA and its twistThe helical twist is one mechanical parameter describing the double helix with a twist of 10.5 base pairs per turn at physiological conditions. Lipfert and his colleagues could prove magnetic tweezers to be a suitable analytical tool applying smaller forces compared to optical tweezers or AFM with the ability for parallel measurement of the twist.Using forces and torques in the piconewton and piconewton nanometer scale, they precisely measured a decrease in helical twist of -11 degrees per kilobasepair DNA and per degree Celsius (i.e. -11.0 ± 1.2 degree/(°C*kbp)) by performing rotational measurements of DNA molecules in a temperature range from 20 to 45 °C, much lower than its melting temperature.

Magnetic tweezers experiments and molecular dynamics simulationsFrom early experiments with circular plasmid DNA in the late 1960’s, it was known that DNA unwinds temperature-dependently. To characterize this effect in detail, Lipfert and his colleagues evolved new techniques.They allow direct measurements of angular changes in DNA twist: “The results of our experimental measurements were consistent with atomistic models showing an almost linear decrease of twist with increasing temperatures.” highlights Dr Franziska Kriegel, first author on the study and former NIM-GP student and now PostDoc in the group of Jan Lipfert. “The magnetic tweezers approach gives us precise measurement results, which in addition can be used to test existing force fields and parameter choices in simulations. The atomistic models deliver insights into structural changes and we found that the unwinding effect is caused by structural changes in the DNA backbone.”

Technically, in magnetic tweezers DNA molecules were tethered between magnetic particles and the flow cell surface. Movable magnets mounted above the flow cell allow the application of calibrated stretching forces and control the twist of the DNA tether by controlling the magnets position in translation and rotation.Using a newly developed objective heating in magnetic tweezers to locally heat DNA molecules up to ~45°C, we were able to precisely measure the change in the DNA’s helical twist: “We could show, that melting of base pairs upon a temperature increase did not contribute to the temperature-dependent DNA twist in the atomistic model simulations.” explains Kriegel.While being a relatively subtle effect, even small changes in DNA twist can play important roles, for example for the pitch and curvature of higher-order DNA origami structures. In addition, the delocalized and temporary base pair breaks revealed by the simulations might well serve as important nucleation points for proteins binding to DNA or for conformational transitions of the DNA helix.